Inhibition of adhesion and migration of an endothelial cell to fibronectin by neutrophil alpha-defensins

ABSTRACT

The present invention relates to the inhibition of angiogenesis by neutrophil alpha-defensins. Further, the present invention relates to methods involving the inhibition of endothelial cell adhesion to the extracellular matrix, endothelial cell apoptosis, and endothelial cell angiogenesis.mediated by alpha-defensins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/983,527, filed Nov. 8, 2004, now abandoned, which claims the benefitof priority pursuant to 35 U.S.C. 119(c) to U.S. Provisional PatentApplication No. 60/518,443, filed on Nov. 7, 2003, all of which areincorporated by reference in their entirety herein.

STATEMENT REGARDING FEDERALLY SUPPORTED RESEARCH OR DEVELOPMENT

This research was supported in part by U.S. Government funds (NationalInstitutes of Health grant HL58107) and the U.S. Government maytherefore have certain rights in the invention.

BACKGROUND OF THE INVENTION

Angiogenesis, the development and growth of new blood vessels, isimportant for organ development, wound healing and various pathologicalconditions such as tumor growth. Angiogenesis involves severalprocesses, such as changes in vascular permeability, as well asendothelial cell adhesion, migration, proliferation and differentiation(Folkman, J. et al., 1996, Cell 87:1153-5). These processes depend bothon a number of growth factors as well as on adhesive contacts with theextracellular matrix (ECM) (Breier, G., A. et al., 1997, Thromb.Haemost. 8:678-683, Risau, W., 1997, Nature 386:671-674, Strombald, S.et al., 1996, Chem. Biol. 3:881-885). ECM-associated proteins, such asfibronectin (FN), vitronectin (VN) and fibrinogen (FBG) are depositedinto an adhesive fibrillar network and control cellular functionincluding growth, differentiation and migration by transmitting signalsto the cells through specific integrins (Giancotti, F. G. et al., 1999,Science 285:1028-32). Observations that mice lacking FN and its receptorα5β1 die early in development and exhibit an improperly formedvasculature indicate that both proteins are crucial participants inphysiologic angiogenesis (Fassler, R. et al., 1995, Genes Dev.9:1896-1908, George, E. L. et al., 1993, Development 119:1079-1091,Yang, J. T. et al., 1993, Development 119:1093-1105). Results of invitro and in vivo studies provide evidence that FN and αβ1 arecritically involved in tumor angiogenesis as well (Hynes, R. O., 2002,Nat. Med. 8:918-921, Kim, S. et al., 2000, Am. J. Pathol.156:1345-1362).

There is emerging evidence that inflammatory cells, and particularlyneutrophils, regulate endothelial cell functions related toangiogenesis. Both pro-angiogenic and anti-angiogenic activities ofneutrophils have been described. Neutrophils are a source of growthfactors such as the vascular endothelial growth factor (VEGF) and matrixmetalloproteinases (Shamamian, P. et al., 2001, J. Cell. Physiol.189:197-206, Lee, S. et al., 2002, J. Clin. Invest. 110:1105-11; Webb,N. J. et al., 1998, Cytokine. 10:254-7). On the other hand,neutrophil-derived elastase can generate the anti-angiogenic factorangiostatin (Scapini, P. et al., 2002, J. Immunol. 168:5798-804). Thenet effect of these and potentially other angiogenic products ofneutrophils may depend on the biological context, but the manner inwhich neutrophils regulate angiogenesis in vivo has not been rigorouslyinvestigated.

α-defensins, a family of four closely related anti-microbial peptides,are the most abundant proteins found in neutrophil granules, comprisingapproximately 5% of the total neutrophil protein content (Ganz, T.2002., J. Clin. Invest. 109:693-697). α-defensins are secreted whenneutrophils are activated during phagocytosis of microorganisms or byspecific exogenous inflammatory agonists. As small cationic peptides,α-defensins can be incorporated into the cell membrane of prokaryoticorganisms during phagocytosis, disrupting ion fluxes and provoking celllysis (Ganz, T. 2002, J. Clin. Invest. 109:693-697, Kagan, B. L. et al.,1994, Toxicology 87:131-149, Ganz, T. et al., 1985, J. Clin. Invest.76:1427-35, Harwig, S. S. et al., 1994, Methods Enzymol. 236:160-172).During severe infections, α-defensins are released into the plasma atconcentrations approaching 30 μM (whereas such concentrations arenormally 15 nM) (Panyutich, A. V. et al., 1993, J. Lab. Clin. Med.122:202-207). α-defensins accumulate in the vessel wall by binding toECM-associated FN (Bdeir, K. et al., 1999, Blood 94:2007-2019) and areabundant in human atherosclerotic plaques (Bdeir, K. et al., 1999, Blood94:2007-2019, Barnathan, E. S. et al., Am. J. Pathol. 150:1009-20); theypromote the accumulation of lipoprotein(a) (Higazi, A. A. et al., 1997,Blood 8 9:4290-4298) and inhibit plasminogen activation (Higazi, A. A.et al, 1996, J. Biol. Chem. 271: 17650-17655).

The role of α-defensins in neutrophil biology has heretofore been poorlyunderstood, for example, with respect to neutrophil biology related totumors. While it is known that tumor vascularization is essential to thegrowth of tumors in a variety of pathophysiological conditions, it isnot known whether α-defensins play any role in neutrophil activityrelated to tumor biology.

Proliferative retinopathies are major causes of blindness. The prominentfeature of these retinopathies is the exuberant neovascularization,which is orchestrated by the hypoxia-induced upregulation of vascularendothelial growth factor (VEGF) that stimulates endothelial cellproliferation, permeability and migration/invasion, as well as by theinteraction of extracellular matrix components like fibronectin (FN)with their integrin receptors.

Pathological neovascularization, for example, is a major cause ofblindness in infancy and during adulthood, complicating such disordersas the retinopathy of prematurity, sickle cell anemia and diabetes,among others. Neutrophil activation is part of each of these processes,but has been heretofore unknown if α-defensin naturally participates incontrolling these processes, or if α-defensin may have any role in thecontrol of these devastating complications.

Because the management and/or inhibition of tumor vascularization is anattractive target for controlling or preventing tumor growth, anunderstanding of the role of neutrophils, and thereby, α-defensins, intumor vascularization and related biology is critical to the developmentof novel anti-angiogenesis therapeutics useful in the treatment ofcancer, as well as other diseases, such as retinopathies associated withexuberant and pathological vascular growth. The present inventionprovides the understanding and goes further to develop therapies andtherefore meets these needs.

SUMMARY OF THE INVENTION

In one embodiment, the present invention features a method of inhibitingadhesion of an endothelial cell to an extracellular matrix (ECM), themethod comprising contacting an endothelial cell with an isolatedα-defensin in an amount sufficient to inhibit adhesion to an ECM. In oneaspect, the method is conducted in vivo. In another aspect, the methodis conducted in vitro.

In one embodiment, a method of the invention includes an α-defensinwhich inhibits the adhesion of said endothelial cell to fibronectin. Inanother embodiment, α-defensin inhibits the migration of an endothelialcell to fibronectin.

In one embodiment, the present invention features a method of inhibitingmigration of an endothelial cell to an ECM, the method comprisingcontacting an endothelial cell with an isolated α-defensin in an amountsufficient to inhibit migration to an ECM.

In an embodiment of the invention, a method of inhibiting adhesion of anendothelial cell to fibronectin comprises contacting an endothelial cellwith an isolated α-defensin in an amount sufficient to inhibit adhesionto fibronectin. In one embodiment, the method is conducted in vivo. Inanother embodiment, the method is conducted in vitro.

In one embodiment of the invention, a method of inhibiting migration ofan endothelial cell to fibronectin comprises contacting an endothelialcell with an isolated α-defensin in an amount sufficient to inhibitmigration to fibronectin. In another embodiment of the invention, amethod of inhibiting adhesion of an endothelial cell to fibronectincomprises contacting fibronectin with an isolated α-defensin in anamount sufficient to inhibit endothelial cell adhesion to fibronectin.In yet another embodiment of the invention, a method of inhibitingmigration of an endothelial cell to fibronectin comprises contactingfibronectin with an isolated α-defensin in an amount sufficient toinhibit migration of an endothelial cell to fibronectin.

In one embodiment, the present invention features a method of inhibitingendothelial cell proliferation, the method comprising contacting anendothelial cell with an isolated α-defensin in an amount sufficient toinhibit endothelial cell proliferation. In another embodiment, theinvention features a method of inducing apoptosis in an endothelialcell, the method comprising contacting an endothelial cell with anisolated α-defensin in an amount sufficient to induce apoptosis in saidcell.

In an embodiment, the invention also features a method of inhibitingangiogenesis in an in vitro system, the method comprising contacting anendothelial cell with an isolated α-defensin in an amount sufficient toinhibit angiogenesis. In another embodiment, the invention features amethod of inhibiting angiogenesis in vivo comprising contactingendothelial cell with an isolated α-defensin in an amount sufficient toinhibit angiogenesis.

In an embodiment of the invention, a method of inhibiting capillary-liketube formation in an in vitro system comprises contacting an endothelialcell with an isolated α-defensin in an amount sufficient to inhibitcapillary-like tube formation. In another embodiment of the invention, amethod of inhibiting capillary formation in vivo comprises contacting anendothelial cell with an isolated α-defensin in an amount sufficient toinhibit capillary formation. In yet another embodiment, the inventionfeatures a method of inhibiting neovascularization comprising contactingendothelial cell with an isolated α-defensin in an amount sufficient toinhibit angiogenesis.

In an embodiment, the invention features a method of modulating abiological condition associated with an endothelial cell in a mammal,the method comprising administering an isolated α-defensin to a mammalin an amount sufficient to modulate a biological condition. Biologicalconditions that can be modulated by a method of the invention include,but are not limited to, vasculogenesis, angiogenesis, vasoregulation,thrombosis homeostasis, diabetic retinopathy, macular degeneration,arthritis, asthma, lung injury, atherosclerosis, and solid tumor cancer.

In another embodiment, the invention features a method of identifying acompound that stimulates angiogenesis in a mammal, the method comprisingcontacting a mammal with a test compound, wherein a higher level ofangiogenesis in the mammal contacted with the test compound comparedwith the level of angiogenesis in a second otherwise identical mammalnot contacted with the test compound is an indication that the testcompound increases the level of angiogenesis in the mammal. In oneaspect, the test compound inhibits the interaction between α-defensinand an endothelial cell.

In an embodiment, the invention features a method of identifying acompound that inhibits angiogenesis in a mammal comprising contacting amammal with a test compound, wherein a lower level of angiogenesis in amammal contacted with the test compound compared with the level ofangiogenesis in a second otherwise identical mammal not contacted withthe test compound is an indication that the test compound decreases thelevel of angiogenesis in the mammal. In one aspect, the test compoundenhances the interaction between α-defensin and an endothelial cell.

In an embodiment, the invention also features a method of treating adisease mediated by hyper-proliferation of endothelial cells in amammal, the method comprising administering to a mammal afflicted with adisease mediated by hyper-proliferation of endothelial cells anendothelial cell-inhibiting amount of an α-defensin.

In one embodiment, the invention further features a kit for modulating abiological process associated with an endothelial cell. Such a kitincludes at least one α-defensin, an applicator, and instructionalmaterial, wherein the instructional material includes instructions forthe use of the kit to modulate a biological process associated with anendothelial cell, and instructions for the use of an α-defensin for thepurpose of modulating a biological process associated with anendothelial cell.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1A is a graph depicting the effect of α-defensin on endothelialcell adhesion. Adhesion of human umbilical vein endothelial cells(HUVEC) to immobilized vitronectin (VN), fibrinogen (FBG) andfibronectin (FN) (each at 5 pg/ml) is shown in the absence (filled bars)or presence of α-defensin (open bars, 5 μM), or a blocking antibodyagainst αvβ3-integrin (for VN and FBG) or against 131-integrin (graybars, antibody concentration 20 μg/ml).

FIG. 1B is a graph depicting the effect of α-defensin on endothelialcell adhesion. Adhesion of HUVEC to immobilized FM (5 μg/ml) is shownalone or in the presence of increasing concentrations of α-defensin, inthe absence (filled squares) or presence of 10 nM Lipoprotein A (opencircles), 20 nM Lipoprotein A (open triangles) or 50 nM Lipoprotein A(filled diamonds). Cell adhesion is expressed as absorbance at 590 nm.Data are shown as the Mean+/−STD (n=3) of a typical experiment; similarresults were obtained in three separate experiments.

FIG. 2A is a graph depicting the effect of α-defensin on endothelialcell migration. Migration of HUVEC towards collagen (CN), vitronectin(YN), fibrinogen (FBG) and fibronectin (FN) is shown alone or after theaddition of VEGF and in the absence (filled bars) or presence ofα-defensin (open bars) (5 μM).

FIG. 2B is a graph depicting the effect of α-defensin on endothelialcell migration. VEGF-stimulated migration of HUVEC towards FN is shownin the absence (−; open bar) or presence (filled bars) of increasingconcentrations of α-defensin, as indicated. Cell migration is expressedas % of control, which is represented as cell migration in the absenceof any stimulus or competitor. Data are Mean+/−STD (n=3) of a typicalexperiment; similar results were obtained in three separate experiments.

FIG. 3A is a graph depicting the effect of α-defensin on endothelialcell proliferation and apoptosis. Proliferation of HUVEC was examined bymeasuring the incorporation of bromodeoxyuridine (BRDU). HUVEC wereincubated with 10 ng/ml VEGF (open bars) or 1 μM sphingosine-1-phosphate(SPP, filled bars) in the absence (−) or presence of increasingconcentrations of α-defensin as indicated. Proliferation of HUVEC isexpressed as % of control, defined as cell proliferation in the absenceof any stimulus or competitor.

FIG. 3B is a graph depicting the effect of α-defensin on endothelialcell proliferation and apoptosis. The apoptosis of HUVEC is shown in theabsence (−; open bar) or presence (filled bars) of increasingconcentrations of α-defensin, of β-defensin (10 μM) or EDTA (5 mM) asindicated. The number of apoptotic cells is expressed as % of the totalcell number. Data are Mean+/−STD (n=3) of a typical experiment; similarresults were obtained in three separate experiments.

FIG. 4A is a collection of images depicting the effect of α-defensin onin vitro capillary-like tube formation. Images of formed tubes in thepresence of 0.1% ECGS and in the absence (“control;” left panel) orpresence of 5 μM α-defensins (“α-defensins;” right panel) are shown.

FIG. 4B is a graph depicting the effect of α-defensin on in vitrocapillary-like tube formation. Capillary-like tube formation wasperformed with bovine retinal endothelial cells (BREC). BREC wereincubated for 48 h with 0.1% ECGS in the absence (−; open bar) orpresence of increasing concentrations of α-defensin (filled bars) orβ-defensin (gray bars), as indicated. Capillary-like tube formation isexpressed as tubes/microcarrier bead. Data are Mean+/−STD (n=3) of atypical experiment; similar results were obtained in three separateexperiments.

FIG. 5 is a collection of images arranged in a grid to illustrate theinhibition of angiogenesis by α-defensin in the chicken chorioallantoicmembrane (CAM)-Assay. Neovascularization in the CAM-Assay was performedas described in the Experimental Examples, in the absence (control) orpresence of α-defensin. The upper left panel depicts a 10× magnificationof the control assay; the upper right panel depicts a 16× magnificationof the control assay; the lower left panel depicts a 10× magnificationof an α-defensin-containing assay; and the lower right panel depicts a16× magnification of an α-defensin-containing assay.

FIG. 6A is a graph depicting the binding of HNPs to α5β1-integrin.¹²⁵I-HNPs in HBS containing 0.1% BSA and 2 mM MnCl₂ was added to wellspre-coated with 2.5 μg/ml α5β1 in HBS/2 mM MnCl₂ for 1 h at 37° C.,washed and the bound radioactivity was counted. Binding to immobilizedBSA was subtracted from total binding as a measure of non-specificbinding. Results from one experiment, representative of three soperformed, is shown. A scatchard plot of the data is shown in theinsert.

FIG. 6B is a graph depicting specific binding of ¹²⁵I-HNPs (1 μM) toimmobilized α5β1-integrin in the absence (−) or presence of RGDS or RGES(1 mg/ml), heparin (4 units/ml), or EDTA (10 mM). Binding to immobilizedBSA was subtracted from total binding as a measure of non-specificbinding.

FIG. 6C is a graph depicting the effect of α-defensin on the binding ofFN to α5β1-integrin. Binding of ¹²⁵I-FN (1 nM) to immobilizedα5β1-integrin (2.5 μg/ml) is shown in the absence or presence ofincreasing concentrations of HNPs as described in panel A. Specificbinding was defined as the difference between binding in the presence of2 mM MnCl₂ and the binding in the presence of 10 mM EDTA which served asnon-specific binding control.

FIG. 6D is a graph depicting the effect of α-defensin Binding of ¹²⁵I-FN(1 nM) to immobilized α5β1-integrin (2.5 μg/ml) or to immobilized BSA isshown in the absence (open bars) or presence of HNPs (filled bars, 5 μM)without (−) or with RGDS or RGES (1 mg/ml), heparin (4 units/ml), orEDTA (10 mM). Total binding is shown. Data are shown as the Mean±STD(n=3) of three separate experiments.

DETAILED DESCRIPTION OF THE INVENTION

Angiogenesis, the growth of new blood vessels, is a complex biologicalprocess that is orchestrated by several growth factors and components ofthe extracellular matrix, including fibronectin (FN) and its receptor,the integrin α5β1. Angiogenesis is a critical part of inflammation andwound repair, but the mechanism by which vascular proliferation andmigration is regulated by inflammatory cells has heretofore beenincompletely understood. As disclosed herein for the first time,α-defensins act as a link between inflammation and angiogenesis. Thepresent invention is based, in part, on the discovery that α5β1-mediatedendothelial cell adhesion and migration to FN is inhibited specificallyand in a dose-dependent manner by α-defensins. The data disclosed hereinalso demonstrate, for the first time, that α-defensins inhibit capillarytube formation in three-dimensional fibrin-matrices. Further, thepresent invention is based, in part, on the discovery that α-defensinsinhibit neovascularization in vivo.

Therefore, in one aspect, the present invention features compositionsand methods comprising α-defensins useful for regulating angiogenesis byaffecting endothelial cell adhesion. The present invention alsofeatures, in another aspect, compositions and methods useful forregulating angiogenesis by affecting endothelial cell migration in aFN-dependent manner. The present invention also features compositionsand methods comprising α-defensins useful for regulating endothelialcell proliferation. Therefore, the present invention provides insightinto the role of inflammatory cells in angiogenesis and provides aplatform for developing a novel anti-angiogenesis drugs, among otherapplications.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization are those well known and commonly employedin the art.

Standard techniques are used for nucleic acid and peptide synthesis. Thetechniques and procedures are generally performed according toconventional methods in the art and various general references (e.g.,Sambrook and Russell, 2001, Molecular Cloning, A Laboratory Approach,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., and Ausubel et al.,2002, Current Protocols in Molecular Biology, John Wiley & Sons, NY),which are provided throughout this document.

The nomenclature used herein and the laboratory procedures used inanalytical chemistry and organic syntheses described below are thosewell known and commonly employed in the art. Standard techniques ormodifications thereof, are used for chemical syntheses and chemicalanalyses.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

As used herein, to “alleviate” a disease, disorder or condition meansreducing the severity of one or more symptoms of the disease, disorderor condition.

By the term “applicator” as the term is used herein, is meant any deviceincluding, but not limited to, a hypodermic syringe, a pipette, abronchoscope, a nebulizer, and the like, for administering the CTHRC1nucleic acid, protein, and/or composition of the invention to a mammal.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the nucleic acid, peptide, and/orcomposition of the invention in the kit for effecting alleviation of thevarious diseases or disorders recited herein. Optionally, oralternately, the instructional material may describe one or more methodsof alleviation the diseases or disorders in a cell or a tissue of amammal. The instructional material of the kit of the invention may, forexample, be affixed to a container which contains the nucleic acid,peptide, and/or composition of the invention or be shipped together witha container which contains the nucleic acid, peptide, and/orcomposition. Alternatively, the instructional material may be shippedseparately from the container with the intention that the instructionalmaterial and the compound be used cooperatively by the recipient.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

A “polynucleotide” means a single strand or parallel and anti-parallelstrands of a nucleic acid. Thus, a polynucleotide may be either asingle-stranded or a double-stranded nucleic acid.

The term “nucleic acid” typically refers to large polynucleotides.

The term “oligonucleotide” typically refers to short polynucleotides,generally, no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences”; sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

A “portion” of a polynucleotide means at least at least about twentysequential nucleotide residues of the polynucleotide. It is understoodthat a portion of a polynucleotide may include every nucleotide residueof the polynucleotide.

A “recombinant polypeptide” is one which is produced upon expression ofa recombinant polynucleotide.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

The term “protein” typically refers to large polypeptides.

The term “peptide” typically refers to short polypeptides.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.

By the term “specifically binds,” as used herein, is meant a compound,e.g., a protein, a nucleic acid, an antibody, and the like, whichrecognizes and binds a specific molecule, but does not substantiallyrecognize or bind other molecules in a sample.

As used herein, to “treat” means reducing the frequency with whichsymptoms of a disease, disorder, or adverse condition, and the like, areexperienced by a patient.

As the term is used herein, “modulation” of a biological process refersto the alteration of the normal course of the biological process. Forexample, modulation of angiogenesis may involve inhibition of theangiogenic process. Alternatively, modulation of angiogenesis mayinvolve stimulation of the angiogenic process. Similarly, “modulation”of any process or interaction is also encompassed by the presentinvention. For example, modulation of the interaction of an endothelialcell with fibronectin may involve inhibition of the interaction of anendothelial cell with fibronectin or it may involve promotion orenhancement of an endothelial cell with fibronectin.

As used herein, the term “extracellular matrix” or “ECM” refers tomaterial surrounding and supporting cells located within mammaliantissues. The ECM may also be referred to as connective tissue, and iscomprised of three major classes of biomolecules, including structuralproteins (e.g., collagen and elastin), specialized proteins (e.g.fibrillin, fibronectin, laminin), and proteoglycans.

The term “α-defensin” refers to any one of human neutrophil peptides 1,2, 3, or 4, and human defensins 5 or 6, also known as HNP-1, HNP-2,HNP-3, HNP-4 HD5, and HD6 respectively.

DESCRIPTION OF THE INVENTION

Methods

A. Methods of Modulating Endothelial Cell Properties and Functions

In one aspect, the present invention features a method of inhibiting abiological interaction of an endothelial cell. In one embodiment, thepresent invention features a method of inhibiting the interaction of anendothelial cell with an extracellular matrix (ECM). As described indetail elsewhere herein, α-defensin is useful to inhibit the adhesion ofan endothelial cell to an ECM. This is because it has been shown hereinfor the first time that α-defensin inhibits that adhesion of anendothelial cell to an ECM.

Therefore, one embodiment of the present invention features a method ofinhibiting endothelial cell adhesion to an ECM by contacting anendothelial cell with α-defensin, wherein the α-defensin inhibitsadhesion of an endothelial cell to an ECM. In another embodiment, amethod of the invention features a method of inhibiting endothelial celladhesion to an ECM by contacting the ECM with α-defensin, wherein theα-defensin inhibits adhesion of the endothelial cell to the ECM. In yetanother embodiment of the invention, adhesion of an endothelial cell toan ECM is inhibited by contacting both the endothelial cell and the ECMwith α-defensin.

As will be understood by the skilled artisan based on the disclosure setforth herein, α-defensin useful in the present invention may be ahomogeneous preparation, or it may be a heterogeneous preparation. Thatis, a single species of α-defensin may be used in a method of thepresent invention. Alternatively, a mixture of two or more differentα-defensins may be used in a method of the present invention. Thepreparation, identification and use of α-defensins of the presentinvention is set forth in detail elsewhere herein.

The present invention also features a method of inhibiting theinteraction of an endothelial cell with fibronectin. This is because ithas been shown herein for the first time that α-defensin inhibitsadhesion of an endothelial cell to fibronectin. In one aspect, a methodof the invention useful for inhibiting the interaction of an endothelialcell with fibronectin comprises contacting an endothelial cell withα-defensin, wherein the α-defensin inhibits adhesion of the endothelialcell to fibronectin. In another embodiment, a method of the inventionuseful for inhibiting the interaction of an endothelial cell withfibronectin comprises contacting fibronectin with α-defensin, whereinthe α-defensin inhibits adhesion of the endothelial cell to fibronectin.In yet another embodiment of the invention, adhesion of an endothelialcell to fibronectin is inhibited by contacting both the endothelial celland fibronectin with α-defensin.

In one aspect, the present invention features a method of inhibiting abiological migration of an endothelial cell. In one embodiment, thepresent invention features a method of inhibiting the migration of anendothelial cell to an extracellular matrix (ECM). As described indetail elsewhere herein, α-defensin is useful to inhibit the migrationof an endothelial cell to an ECM. This is because it has been shownherein for the first time that α-defensin inhibits the migration of anendothelial cell to an ECM.

Therefore, one embodiment of the present invention features a method ofinhibiting endothelial cell migration to an ECM by contacting anendothelial cell with α-defensin, wherein the α-defensin inhibitsmigration of an endothelial cell to an ECM. In another embodiment, amethod of the invention features a method of inhibiting endothelial cellmigration to an ECM by contacting the ECM with α-defensin, wherein theα-defensin inhibits migration of the endothelial cell to the ECM. In yetanother embodiment of the invention, migration of an endothelial cell toan ECM is inhibited by contacting both the endothelial cell and the ECMwith α-defensin.

The present invention also features a method of inhibiting thebiological migration of an endothelial cell to fibronectin. This isbecause it has been shown herein for the first time that α-defensininhibits migration of an endothelial cell to fibronectin. In one aspect,a method of the invention useful for inhibiting the migration of anendothelial cell to fibronectin comprises contacting an endothelial cellwith α-defensin, wherein the α-defensin inhibits migration of theendothelial cell to fibronectin. In another embodiment, a method of theinvention useful for inhibiting the migration of an endothelial cell tofibronectin comprises contacting fibronectin with α-defensin, whereinthe α-defensin inhibits migration of the endothelial cell tofibronectin. In yet another embodiment of the invention, migration of anendothelial cell to fibronectin is inhibited by contacting both theendothelial cell and fibronectin with α-defensin.

In one aspect, the present invention features a method of inducing orstimulating a biological process of an endothelial cell. In oneembodiment, the present invention features a method of inducingapoptosis of an endothelial cell. As described in detail elsewhereherein, α-defensin is useful to induce apoptosis of an endothelial cell.This is because it has been shown herein for the first time thatα-defensin induces apoptosis in an endothelial cell.

Therefore, one embodiment of the present invention features a method ofinducing endothelial cell apoptosis by contacting an endothelial cellwith α-defensin, wherein the α-defensin induces apoptosis of theendothelial cell. In another embodiment, the invention features a methodof inducing apoptosis of an endothelial cell by contacting an ECM withα-defensin, wherein interaction between the endothelial cell and the ECMprovides α-defensin to the endothelial cell, and the α-defensin therebyinduces apoptosis of the endothelial cell. In yet another embodiment ofthe invention, apoptosis of an endothelial cell is induced by contactingboth the endothelial cell and the ECM with α-defensin.

In another embodiment of the present invention is provided a method ofinducing apoptosis of an endothelial cell by contacting fibronectin withα-defensin, wherein interaction between the endothelial cell andfibronectin provides α-defensin to the endothelial cell, and theα-defensin thereby induces apoptosis of the endothelial cell. In yetanother embodiment of the invention, apoptosis of an endothelial cell isinduced by contacting both the endothelial cell and fibronectin withα-defensin.

In another aspect, the present invention features a method of inhibitinga biological process of an endothelial cell. In one embodiment, thepresent invention features a method of inhibiting the proliferation ofan endothelial cell. As described in detail elsewhere herein, α-defensinis useful to inhibit the proliferation of an endothelial cell. This isbecause it has been shown herein for the first time that α-defensininhibits proliferation of an endothelial cell.

Therefore, one embodiment of the present invention features a method ofinhibiting endothelial cell proliferation by contacting an endothelialcell with an α-defensin, wherein the α-defensin inhibits proliferationof the endothelial cell. In another embodiment, the invention features amethod of inhibiting proliferation of an endothelial cell by contactingan ECM with α-defensin, wherein interaction between the endothelial celland the ECM provides α-defensin to the endothelial cell, and theα-defensin thereby inhibits proliferation of the endothelial cell. Inyet another embodiment of the invention, proliferation of an endothelialcell is inhibited by contacting both the endothelial cell and the ECMwith α-defensin.

In another embodiment of the present invention is provided a method ofinhibiting proliferation of an endothelial cell by contactingfibronectin with α-defensin, wherein interaction between the endothelialcell and fibronectin provides α-defensin to the endothelial cell, andthe α-defensin thereby inhibits proliferation of the endothelial cell.In yet another embodiment of the invention, inhibition of theproliferation of an endothelial cell is induced by contacting both theendothelial cell and fibronectin with α-defensin.

The present invention also features a method of modulating processes andfunctions associated with endothelial cells. As described in detailelsewhere herein, in vitro and in vivo processes and functionsassociated with endothelial cells can be modulated by α-defensin. In oneaspect of the invention, a method of inhibiting an endothelial cellprocess or function in vitro is provided.

In one embodiment, a method of inhibiting an endothelial cell process orfunction in vitro includes contacting an endothelial cell withα-defensin, whereby the α-defensin inhibits a process or a functionassociated with the endothelial cell. In one aspect, a method of theinvention is useful for inhibition of angiogenesis in vitro. In anotheraspect, a method of the invention is useful for inhibition ofcapillary-like tube formation in vitro.

Another aspect of the invention provides a method for inhibitingmigration of an endothelial cell to an ECM. In still another aspect, amethod of the invention is useful for inhibition of migration of anendothelial cell to fibronectin. In another aspect of the invention, amethod is provided for inhibition of the adhesion of an endothelial cellto an ECM. In still another aspect, a method is provided for theinhibition of an endothelial cell to fibronectin.

The present invention also features a method of inhibiting processes andfunctions associated with endothelial cells in vivo. In one aspect ofthe invention, a method of inhibiting a process or function of anendothelial cell in vivo includes contacting an endothelial cell withα-defensin, whereby the α-defensin inhibits a process or a functionassociated with the endothelial cell in vivo. In one embodiment, amethod of the invention inhibits angiogenesis in vivo. In anotherembodiment, a method of the invention inhibits capillary-like tubeformation in vivo. In yet another embodiment, a method of the inventioninhibits neovascularization in vivo.

The present invention also features a method of inhibiting anendothelial cell-associated biological process in a living organism. Asdescribed in detail elsewhere herein, α-defensins have been shown by wayof the present invention to possess anti-angiogenic activity in chicken.That is, data presented herein demonstrate that angiogenesis isinhibited in a living organism upon contacting the organism withα-defensin. As described herein, such inhibition occurs at or near thelocation of contact with α-defensin.

As set forth elsewhere herein for the first time, the ability ofα-defensin to inhibit endothelial cell migration to ECM and fibronectin,the ability of α-defensin to inhibit endothelial cell adhesion to ECMand fibronectin, and the ability of α-defensin to inhibit endothelialcell proliferation and to induce endothelial cell apoptosis all conferanti-angiogenic or anti-vascularization properties upon α-defensin. Inparticular, the properties of α-defensin disclosed herein for the firsttime demonstrate the roles of α-defensin in biological processes relatedto inflammation, wound healing, vascularization, angiogenesis, diabeticretinopathy, macular degeneration, arthritis, asthma, lung injury,atherosclerosis, exuberant angiogenesis leading to blindness, and solidtumors, among others.

Therefore, the present invention includes methods of modulating abiological process using α-defensin. Based on the disclosure providedherein for the first time, one of skill in the art will understand thatany biological process in which α-defensin plays a role is a biologicalprocess that may be modulated by α-defensin according to the presentinvention.

One example of the modulation of a biological process according to thepresent invention includes the treatment of a patient that may benefitfrom modulation of an endothelial cell-related biological process by wayof an α-defensin-based method or composition, which compositions areaddressed elsewhere herein. By way of a non-limiting example, a methodof the present invention is useful to treat a patient that can benefitfrom inhibition of angiogenesis. As set forth in detail elsewhereherein, it has been found that α-defensin can be used to inhibitangiogenic properties of an endothelial cell, including, but not limitedto endothelial cell migration to an ECM, endothelial cell migration tofibronectin, and endothelial cell proliferation. Therefore, in oneembodiment, the present invention provides a method of inhibitingangiogenesis in a patient in need thereof by contacting an endothelialcell of the patient with at least one α-defensin.

It will be understood by the skilled artisan, when equipped with thepresent disclosure, that methods set forth herein that are useful invivo are equally applicable to the treatment of a patient that maybenefit from such treatment.

In another aspect of the present invention, a method of inhibiting abiological process using α-defensin is provided. In one embodiment, theinvention features a method of inhibiting vasculogenesis. A method ofinhibiting vasculogenesis according to the present invention includescontacting an endothelial cell with α-defensin, whereby vasculogenesisis inhibited. The present invention also features a method of inhibitingneovascularization. In one embodiment, a method of inhibitingneovascularization includes contacting an endothelial cell withα-defensin, whereby neovascularization is inhibited.

In another embodiment, the invention features a method of inhibitingangiogenesis. A method of inhibiting angiogenesis according to thepresent invention includes contacting an endothelial cell withα-defensin, whereby angiogenesis is inhibited. In one aspect, a methodof inhibiting angiogenesis includes inhibiting exuberant angiogenesisthat leads to blindness. Further still, the present invention alsofeatures a method of inhibiting angiogenesis at a site of inflammation.In one embodiment, a method of inhibiting angiogenesis at a site ofinflammation includes contacting an endothelial cell with α-defensin,thereby inhibiting angiogenesis at a site of inflammation.

As described elsewhere herein, contacting an ECM or fibronectin withα-defensin is also useful in methods of the present invention. By way ofa non-limiting example, a method including contacting an ECM orfibronectin with an α-defensin may be used to inhibit angiogenesis,wherein the ECM or fibronectin provides α-defensin to an endothelialcell, thereby inhibiting angiogenesis.

Therefore, the present invention also features a method of modulating abiological process using α-defensin, wherein the method includescontacting an ECM or fibronectin with α-defensin. Based on thedisclosure provided herein for the first time, one of skill in the artwill understand that any biological process in which α-defensin plays arole is a biological process that may be modulated by contacting an ECMor fibronectin α-defensin according to the present invention.

In one aspect of the present invention is provided a method ofinhibiting a biological process by contacting an ECM or fibronectin withα-defensin. In one embodiment, the invention features a method ofinhibiting vasculogenesis. A method of inhibiting vasculogenesisaccording to the present invention includes contacting an ECM orfibronectin with α-defensin, whereby the ECM or fibronectin providesα-defensin to an endothelial cell, thereby inhibiting vasculogenesis.The present invention also features a method of inhibitingneovascularization. In one embodiment, a method of inhibitingneovascularization according to the present invention includescontacting an ECM or fibronectin with α-defensin, whereby the ECM orfibronectin provides α-defensin to an endothelial cell, therebyinhibiting neovascularization.

In another embodiment, the invention features a method of inhibitingangiogenesis. A method of inhibiting angiogenesis according to thepresent invention includes contacting an ECM or fibronectin withα-defensin, whereby the ECM or fibronectin provides α-defensin to anendothelial cell, thereby inhibiting angiogenesis. Further still, thepresent invention also features a method of inhibiting angiogenesis at asite of inflammation. In one embodiment, a method of inhibitingangiogenesis at a site of inflammation according to the presentinvention includes contacting an ECM or fibronectin with α-defensin,whereby the ECM or fibronectin provides α-defensin to an endothelialcell, thereby inhibiting angiogenesis at a site of inflammation.

The present invention also features methods of using α-defensin tomodulate other biological processes associated with an endothelial cell.Such processes include, but are not limited to, vasoregulation, andthrombosis homeostasis, as described in detail elsewhere herein.

In one aspect of the invention, modulation of a biological processoccurs in vitro. In another aspect, modulation of a biological processoccurs in vivo. The present invention also features compositions andmethods useful for the treatment of a living organism. In oneembodiment, the living organism is a chicken. In another embodiment, theliving organism is a mammal, including, but not limited to a human.

One skilled in the art would understand, based upon the disclosureprovided herein, that since inhibiting endothelial cell migration and/oradhesion to an ECM, and in particular, to fibronectin, can mediate abeneficial effect, methods of inhibiting migration and/or adhesion of anendothelial cell to an ECM or to fibronectin can be used to treat and/oralleviate a biological process, disease, disorder or conditionassociated with an endothelial cell in an organism, where a higher levelof biological function of an endothelial cell prior to, or in theabsence of, treatment provides a benefit. A benefit such as preventingundesired angiogenesis or vascularization is disclosed elsewhere herein.

Thus, whether an α-defensin is administered by contacting an endothelialcell, an ECM, or fibronectin, the present invention includes a methodwhere a biological function of an endothelial cell is modulated in orderto treat/or alleviate a disorder associated with endothelial cellbiology. Such treatment may include, but is not limited to, inhibitionof angiogenesis, inhibition of neovascularization, inhibition ofvasculogenesis, and the like. The skilled artisan will know, based onthe disclosure provided herein, that an endothelial cell-basedbiological process that can be modulated by α-defensin is encompassed bythe present invention.

The present invention also features a method of inhibiting pathologicalretinal angiogenesis in a mammal. This is because it has been shownherein for the first time that administration of α-defensin results inabout 40% reduction of retinal neovascularization. As described indetail elsewhere herein, α-defensin reduces both the rapid as well asthe delayed VEGF-induced increase in endothelial permeability. Moreover,α-defensin inhibits VEGF-induced proliferation of BREC in adose-dependent manner, as well as capillary tube formation inthree-dimensional fibrin-matrices.

Therefore, in one embodiment, the present invention features a method ofinhibiting pathological retinal neovascularization in a mammal. Themethod includes contacting the retinal membrane of a mammal with anα-defensin in order to inhibit retinal neovascularization. Methods oftreating a mammal according to the present invention are set forth indetail elsewhere herein.

B. Methods of Identifying Useful Compounds

The present invention further includes a method of identifying acompound that modulates a biological process in a mammal. The methodcomprises contacting a mammal with a test compound, wherein a higher orlower level of the biological process in a mammal contacted with thetest compound compared with the level of the biological process in asecond otherwise identical mammal not contacted with the test compoundis an indication that the test compound modulates the level of thebiological process in the mammal contacted with the compound. If thetest compound is additionally found to modulate the interaction betweenα-defensin and an endothelial cell, then the test compound is therebyidentified as a compound that stimulates angiogenesis in a cell.

Methods of identifying an increase or decrease in a biological processthat can be modulated using a method or composition of the presentinvention are set forth in detail elsewhere herein. Additional methodsuseful for identifying an increase or a decrease in a biological processwill be apparent to the skilled artisan in view of the presentdisclosure, and will therefore not be discussed in detail. Methods foridentifying interaction between a polypeptide and a cell, such asbetween an α-defensin and an endothelial cell, are also well-known inthe art, and will not be discussed in detail.

The present invention features a method of identifying a compound thatstimulates angiogenesis in a mammal. The method comprises contacting amammal with a test compound, wherein a higher level of angiogenesis in amammal contacted with the test compound compared with the level ofangiogenesis in a second otherwise identical mammal not contacted withthe test compound is an indication that the test compound increases thelevel of angiogenesis in the mammal contacted with the compound. If thetest compound is also found to inhibit the interaction betweenα-defensin and an endothelial cell, then the test compound is therebyidentified as a compound of the present invention that stimulatesangiogenesis in a cell.

The invention also includes a method of identifying a compound thatinhibits angiogenesis in a mammal. The method comprises contacting amammal with a test compound, wherein a lower level of angiogenesis in amammal contacted with the test compound compared with the level ofangiogenesis in a second otherwise identical mammal not contacted withthe test compound is an indication that the test compound decreases thelevel of angiogenesis in the mammal contacted with the compound. If thetest compound is also found to enhance or stabilize the interactionbetween α-defensin and an endothelial cell, then the test compound isthereby identified as a compound of the present invention that inhibitsangiogenesis in a cell.

Numerous methods of measuring angiogenesis in a mammal are known in theart, and will therefore not be described herein. Briefly, angiogenesiscan be measured in a mammal using a method such as, but not limited to,

The invention also features methods of modulating other biologicalprocesses, diseases, disorders and the like. Other biologicalprocesses/conditions that can be modulated by methods and compositionsof the present invention include, but are not limited to,vasculogenesis, neovascularization, vasoregulation, thrombosishomeostasis, diabetic retinopathy, arthritis, asthma, lung injury,atherosclerosis, and solid tumors.

Compositions

The invention includes a composition comprising an isolated α-defensin.In one aspect of the invention, the composition comprises apharmaceutically acceptable carrier.

The compositions can be used to administer α-defensin to a cell, atissue, or an animal. The compositions are useful to treat a disease,disorder or condition mediated by an endothelial cell such thatdecreasing or increasing a biological process of an endothelial cell ina tissue or animal is beneficial to the animal. That is, where adisease, disorder or condition in an animal is mediated by or associatedwith the normal or altered biological activity of an endothelial cell, acomposition of the present invention can be used to modulate suchactivity of an endothelial cell.

For administration to a mammal, a polypeptide can be suspended in anypharmaceutically acceptable carrier, for example, HEPES buffered salineat a pH of about 7.8.

Other pharmaceutically acceptable carriers which are useful include, butare not limited to, glycerol, water, saline, ethanol and otherpharmaceutically acceptable salt solutions such as phosphates and saltsof organic acids. Examples of these and other pharmaceuticallyacceptable carriers are described in Remington's Pharmaceutical Sciences(1991, Mack Publication Co., New Jersey).

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered, prepared, packaged, and/or sold informulations suitable for oral, rectal, vaginal, parenteral, topical,pulmonary, intranasal, buccal, ophthalmic, or another route ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations.

The compositions of the invention may be administered via numerousroutes, including, but not limited to, oral, rectal, vaginal,parenteral, topical, pulmonary, intranasal, buccal, or ophthalmicadministration routes. The route(s) of administration will be readilyapparent to the skilled artisan and will depend upon any number offactors including the type and severity of the disease or conditionbeing treated, the type and age of the veterinary or human patient beingtreated, and the like.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in oral solid formulations,ophthalmic, suppository, aerosol, topical or other similar formulations.In addition to the compound such as heparan sulfate, or a biologicalequivalent thereof, such pharmaceutical compositions may containpharmaceutically-acceptable carriers and other ingredients known toenhance and facilitate drug administration. Other possible formulations,such as nanoparticles, liposomes, resealed erythrocytes, andimmunologically based systems may also be used to administer α-defensinaccording to the methods of the invention.

Compounds which are identified using any of the methods described hereinmay be formulated and administered to a mammal for treatment ofexcessive or insufficient angiogenesis, vasculogenesis, vasoregulation,thrombosis homeostasis, and the like, are now described. As described indetail elsewhere herein, such a compound may be an α-defensin, oralternatively, such a compound may be a non-α-defensin, wherein thecompound has been demonstrated by a method set forth herein to modulatethe interaction between an α-defensin and an endothelial cell, anendothelial cell and an ECM, an endothelial cell and fibronectin, andthe like.

The invention encompasses the preparation and use of pharmaceuticalcompositions comprising a compound useful for treatment of excessive orinsufficient angiogenesis, vasculogenesis, vasoregulation, thrombosishomeostasis, and the like, as an active ingredient. Such apharmaceutical composition may consist of the active ingredient alone,in a form suitable for administration to a subject, or thepharmaceutical composition may comprise the active ingredient and one ormore pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, ophthalmic, intrathecal or another route of administration.Other contemplated formulations include projected nanoparticles,liposomal preparations, resealed erythrocytes containing the activeingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.,polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e., about 20° C.) and which isliquid at the rectal temperature of the subject (i.e., about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition may be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or gel or cream or a solution for vaginalirrigation.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made bycombining the active ingredient with a pharmaceutically acceptableliquid carrier. As is well known in the art, douche preparations may beadministered using, and may be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject. Douche preparations mayfurther comprise various additional ingredients including, but notlimited to, antioxidants, antibiotics, antifungal agents, andpreservatives.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen-free water) prior toparenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and preferably from about 1 toabout 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken, i.e., by rapid inhalation throughthe nasal passage from a container of the powder held close to thenares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 nanometers, and may further comprise one or moreof the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other ophthalmologically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed. (1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa.), which isincorporated herein by reference.

Typically, dosages of the compound of the invention which may beadministered to an animal, preferably a human, will vary depending uponany number of factors, including but not limited to, the type of animaland type of disease state being treated, the age of the animal and theroute of administration.

The compound can be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even leesfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, etc.

V. Kits

The invention includes various kits which comprise a compound, such asan α-defensin or a mixture of α-defensins. Although exemplary kits aredescribed below, the contents of other useful kits will be apparent tothe skilled artisan in light of the present disclosure. Each of thesekits is included within the invention.

In one aspect, the invention includes a kit for alleviating a disease,disorder or condition mediated by an endothelial cell. The kit is usedpursuant to the methods disclosed in the invention. Briefly, the kit maybe used to contact an endothelial cell with α-defensin, wherein theaction of α-defensin on the endothelial cell mediates a beneficialeffect.

The kit further comprises an applicator useful for introducingα-defensin to the endothelial cell. The particular applicator includedin the kit will depend on, e.g., the form of α-defensin and/or thecomposition used to introduce α-defensin to the cell. Such applicatorsare well-known in the art and may include, among other things, animplant, a syringe, and the like. Moreover, the kit comprises aninstructional material for the use of the kit. These instructions simplyembody the disclosure provided herein. The kit may also include apharmaceutically-acceptable carrier. The composition is provided in anappropriate amount as set forth elsewhere herein. Further, the route ofadministration and the frequency of administration are as previously setforth elsewhere herein.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Materials and Methods

Reagents: α-defensins (human neutrophil peptides 1-3) were isolated fromhuman Neutrophils and from sputum of cystic fibrosis patients andcharacterized as described by Harwig et al. (1994, Methods Enzymol.236-:160-172). Three bands of HNP1-3 were detected on SDS-PAGE bywestern blot and commassie staining. A murine monoclonal antibodyagainst human α-defensin was used. Lipoprotein (a) [Lp(a)] was isolatedusing lysinesepharose chromatography as described by Bdeir et al. (1999,Blood 94:2007-2019). Recombinant human β-defensins (HBD-2) were alsoused. VN was purified from human plasma and converted to the multimericform as described by Chavakis et al. (1998, Blood 91:2305-2312); FBG, FNand sphingosine-1-phosphate were purchased from Sigma (Munich, Germany);monoclonal antibody against β-1 integrin was from DAKO (Hamburg,Germany), cyclic RGDfV peptide was from Bachem (Heidelberg, Germany).Monoclonal antibody against β-1 was also used. Endothelial cell growthserum (ECGS) was from PromoCell (Heidelberg, Germany). Basic fibroblastgrowth factor (bFGF) and vascular endothelial growth factor (VEGF) werefrom R&D Systems (Wiesbaden, Germany).

Cell culture: Bovine retinal endothelial cells (BREC) were used andcultivated and human umbilical vein endothelial cells (HUVEC) asdescribed by. All culture media were from Gibco (Eggenstein, Germany).

Cell adhesion assays: Cell adhesion to multiwell plates coated with FN,YN and FBG (and to BSA-coated wells as control) was tested according topreviously described protocols (Chavakis et al., 2000, Blood96:514-522). Briefly, multiwell plates were coated with 5 μg/ml FN, YNor FBG (dissolved in bicarbonate buffer, pH 9.6), respectively, andblocked with 3% (wt/vol) BSA. Confluent BREC or HUVEC were detached withtrypsin, which was subsequently neutralized with soybean trypsininhibitor (Sigma, Germany), were washed in serum-free DMEM and platedonto the precoated wells at 37° C. in the absence or presence ofcompetitors in serum-free DMEM. After an incubation period of 60 mm thewells were washed and the number of adherent cells and the intensity ofstaining with crystal violet was quantified at 590 nm.

Cell migration assays: Chemotaxis of HUVEC and BREC was tested inmodified Boyden chamber assays. Polycarbonate filter membranes(Nucleopore; Whatman, Clifton, N.J.) with 8 μm pore size were coatedwith FN, VN, FBG or collagen 1(5 μg/ml) in PBS for 16 hours at 4° C. andthen washed and air dried before use. After gentle trypsinization (seeabove), the cells were resuspended in DMEM contains 0.2% (vol/vol) FCS.Each factor was tested in triplicates using 20,000 cells in the upperwell, with the test factors in the lower wells. After 3 hours at 37° C.,the upper side of the membrane was scraped with a cell scraper to removenon-adherent cells. Membranes were fixed with methanol-acetone (1:1) for30 mm and stained with crystal violet for another 30 min, then washed inwater and fixed between two slides. Adherent cells were counted and adensitometric analysis of the stained spots was made using the Scionimage software (NIH, Bethesda, Md.).

Cell proliferation: Endothelial cell proliferation was determined bymeasuring the incorporation of bromodeoxyuridine (BrdU). Endothelialcells were plated onto 96-well plates and incubated for 12 hours afterwhich the medium was changed to MCDB-131 containing 0.05% FCS. Cellswere then incubated for 24 hours in the absence or presence of stimulior competitors. BrdU was added to the medium during the last 6 hours ofincubation. The cells were then fixed and cell proliferation wasquantified with a standardized calorimetric immunoassay (RocheDiagnostics, Mannheim, Germany) according to the manufacturer'sinstructions.

Detection of apoptosis using flow cytometry: Endothelial cell apoptosiswas measured as described by Al-Fakhri et al. (2003, Biol. Chem.384:423-435). HUVEC or vascular smooth muscle cells were cultivated for16 hours in the absence or presence of stimuli or competitors;thereafter, cells were detached with trypsin (2.5 mg/ml) that wasimmediately neutralized with soybean trypsin inhibitor. The cells wereharvested by pelleting at 500 g/5 mm, washed repeatedly with PBS, pH7.5, and then resuspended in ice-cold binding buffer (10 mmol/lHEPES/NaOH, pH 7.5, 140 mmol/l NaCl, 2.5 mmol/l CaCl₂). The percentageof apoptotic cells was quantified using the Annexin V-FITC kit(Immunotech/Beckman Coulter, Marseille, France). Counterstaining withpropidium iodide (red fluorescence) was performed to discriminatebetween necrotic and apoptotic cells. Briefly, cells were incubated for10 mm at RT with 0.5 μg/ml FITC-labeled annexin V and 2 μg/ml propidiumiodide, and then washed with binding buffer to avoid induction ofapoptosis through the staining process. FACS analysis (Epics-XL, BeckmanCoulter Electronics, Krefeld, Germany) was immediately carried out usingstandard protocols. Apoptotic cells were defined as those that exhibitedexclusively green fluorescent signals within pre-defined gating criteriaof forward and sideward scatter. The maximum rate of apoptosis inductionfor a 16 hours-cultivation period had been determined in initialexperiments.

Capillary tube formation: A protocol described by Koblizek et al. (1998,Curr. Biol. 8:529-532) was modified to use BREC grown on Cytodex-3microcarriers (Sigma; St. Louis, Mo.) at 37° C. with 5% CO₂ in completeMCDB-1 31 medium. Approximately 100 confluent microcarriers coated withBREC were added per well to sterile-filtered solutions containing 1.8mg/ml fibrinogen in PBS and then α-thrombin (0.65 NIH U/ml) wasimmediately added for 30 mm to induce fibrin clot formation. Then, 1 mlof medium containing 200 KIU/ml aprotinin alone or together with 0.1%ECGS and in the absence or presence of competitors was added and plateswere incubated for an additional 48 hours at 37° C. The number ofcapillary-like sprouts was evaluated microscopically and was expressedas sprouts/microcarrier.

Chicken chorioallantoic membrane (CAM) angiogenesis assay: The CAM assaywas performed to determine the anti-angiogenic effect of α-defensin invivo using a previously described method of Ausprunk et al. (1975, Am.J. Pathol. 79:597-628), with minor modifications. Fertilized chickeneggs were prepared by cutting a window into the shell on day 3 ofincubation at 37° C. in a humidified incubator. On day 10,methylcellulose disks saturated with α-defensin (100 μM or 300 μM), orwith bFGF (50 ng) as a positive control, or with buffer alone as anegative control were laid onto the egg chorionallantoic membrane. Bloodvessel density around (magnification: ×10) or within the disks(magnification: ×16) was evaluated and photographed using astereomicroscope (Leica, Wetzlar, Germany) on day 13. Five CAMs wereanalyzed for each test group and the experiments were repeated at leastthree times.

Example 1 Inhibition of Endothelial Cell Adhesion and Migration byα-defensins

Endothelial cells adhere via different integrins to matrix proteins suchas FN, FBG, or VN. Whereas endothelial cell adhesion to FBG and VN ispredominantly dependent on αv-integrins, β1-integrins mediate adhesionto FN (FIG. 1A). The effect of α-defensins on the adhesion of HUVEC andBREC to these different matrix proteins was investigated. α-defensinsspecifically blocked endothelial cell adhesion to FN, whereas adhesionto FBG or VN was not affected (FIG. 1A). The anti-adhesive effect ofα-defensins was dose-dependent (FIG. 1B), reaching a maximum of ˜90% atα-defensin concentration of 10 μM. well within the plasma concentrationdetected in systemic infection. As α-defensins promote the binding ofLp(a) to FN in the extracellular matrix of vascular cells, the effect ofLp(a) on the antiadhesive activity of α-defensins was tested. Lp(a) didnot affect endothelial cell adhesion to any of the substrates directlyand the antiadhesive effect of α-defensins on FN-adhesion was notaltered in the presence of Lp(a) (FIG. 1B).

α-defensins also specifically reduced the migration of HUVEC towardsfibronectin both under control conditions and under stimulation withVEGF, whereas migration towards collagen, VN or FBG was not affected(FIG. 2A). Again, inhibition of HUVEC migration towards FM byα-defensins was dose-dependent (FIG. 2B) and not affected by Lp(a) (notshown). Similar results were observed with BREC. Neither endothelialcell adhesion to FN nor migration towards FN were affected byβ-defensins even at the highest concentrations tested (10 μM). Theseresults demonstrate that α-defensins block endothelial cell adhesion andmigration in a α5β1-integrin-FN-specific manner.

Example 2 Inhibition of Endothelial Proliferation by α-defensins

Endothelial cell proliferation is stimulated during angiogenesis.Therefore, the influence of α-defensins on HUVEC proliferation wasexamined by measuring de novo DNA synthesis by measuring theincorporation of BrdU. VEGF and sphingosine-1-phosphate are knownactivators of endothelial cell proliferation. Both agonists increasedDNA synthesis approximately two-fold over a period of 24 hours. HUVECproliferation was inhibited in the presence of α-defensins in adose-dependent manner both under control conditions as well as understimulation with VEGF or sphingosine-1-phosphate (FIG. 3A). To elucidatethe mechanism by which α-defensins inhibit proliferation, the inductionof apoptosis by α-defensins was examined. α-defensins induced theapoptosis of HUVEC cultivated on FN-coated plates in a dose-dependentmanner (FIG. 3B). In contrast, β-defensins did not influence HUVECproliferation or apoptosis at the same concentrations (FIG. 3B).α-defensins did not affect the proliferation or apoptosis of other celltypes tested, such as smooth muscle cells and fibroblasts at theseconcentrations, nor did Lp(a) affect the anti-proliferative and thepro-apoptotic effect of α-defensin. Together, these studies demonstratethat α-defensins specifically induce apoptosis of endothelial cells andthereby inhibit endothelial cell proliferation.

Example 3 Inhibition of Capillary Sprout Formation by α-defensins

The data set forth herein demonstrate that α-defensins can regulateendothelial cell adhesion, migration and proliferation in vitro, allthree of which processes essential for neovascularization. Therefore,the role of α-defensins was examined in a more complex“angiogenesis-resembling” in vitro assay that simulated angiogenesis.Capillary-like tube formation in three-dimensional fibrin gels dependson vascular permeability as well as on the invasive, migratory andproliferative potential of endothelial cells. In this system,α-defensins reduced the number of capillary-like tubes formed during anincubation period of 48 hours in the presence of 0.1% of ECGS. Theextent of inhibition was dose-dependent and mirrored the observed invitro activity; almost complete inhibition of tube formation byα-defensins was observed at concentrations above 5 μM (FIGS. 4A and 4B).Again, β-defensins had no effect on capillary tube formation in thisexperimental setting.

Example 4 Inhibition of Angiogenesis in the CAM-Assay by α-defensins

The anti-angiogenic activity of α-defensins in vivo was investigatedusing the CAM assay, as described elsewhere herein. On day 13, there wasa virtual absence of capillaries at center of the methylcellulose discand the fragmentation of preexisting small vessels in CAMs exposed toα-defensins, while flanking larger vessels were unaffected (FIG. 5,magnification: left panels, ×10; right panels, ×16). α-defensinsinhibited angiogenesis both under control conditions and understimulation with bFGF. In control experiments, buffer alone had noeffect on vessel branching (FIG. 5).

Example 5 Measurement of Angiogenesis in a Mammal

Angiogenesis was measured in a mammal using a MATRIGEL assay(Collaborative Biomedical Products; Bedford, Mass.). Several sampleswere prepared, including MATRIGEL alone, MATRIGEL containing growthfactor bFGF, and MATRIGEL mixed with 1×10⁶/ml B16/F10 melanoma cells.Samples were injected subcutaneously in both flanks of 8 week-old C57B16mice. In one flank, the tested compound was added; in the other flank,buffer was used as the control. Five days later, the MATRIGEL pellet wasexposed. Blood vessel density around or within the MATRIGEL wasquantified.

Angiogenesis was also measured in a mammal using a cornea micropocketassay, as described by Zhou et al. (2004, Cancer Res. 64:4699-4702).Briefly, a corneal micropocket is created on one eye of a 6-week oldmouse. A micropellet containing bFGf is implanted, and cornealmicrovascularization was examined 5 days later (DeLisser et al., 1997,Am. J. Pathol. 151:671-677).

Example 6 Inhibition of Pathological Retinal Neovascularization byα-defensin

The effect of α-defensins on pathological retinal angiogenesis wasexamined. In vitro, α5β1-integrin-dependent migration of bovine retinalendothelial cells (BREC) to FN, both under control conditions and understimulation by VEGF, was inhibited specifically by α-defensins. Inaddition, α-defensins reduced both the rapid (1 h), as well as thedelayed VEGF-induced increase in endothelial permeability. Moreover,α-defensins inhibited VEGF-induced proliferation of BREC in adose-dependent manner, as well as capillary tube formation inthree-dimensional fibrin-matrices. In vivo, an upregulation ofα5β1-integrin and FN was observed in the mouse model of hypoxia-inducedretina angiogenesis, as compared to normal retinas. In this model,administration of α-defensins resulted in about 40% reduction of retinalneovascularization. Accordingly, higher apoptosis was observed inretinas of mice that were treated with α-defensins as compared tocontrol-treated mice. Taken together, these results indicate thatα-defensins can inhibit pathological retinal neovascularization in vivoand may provide the platform for developing a clinically feasible andefficient strategy against proliferative retinopathies.

Emerging evidence points to the likelihood that inflammatory changeswithin the vessel wall regulate the neovascularization that isassociated with tumor growth, wound healing and inflammatory diseasessuch as psoriasis and atherosclerosis. Inflammatory cells such asneutrophils, recruited to the inflamed or injured tissue, can releasegrowth factors as well as proteases capable of modulating tissuestructure and promoting angiogenesis. On the other hand,neutrophil-derived elastase can generate the anti-angiogenic factorangiostatin and the net effect of neutrophil products on the angiogenicprocess is unknown.

In the present application, the effect of α-defensins, the most abundantproteins secreted by activated neutrophils, on angiogenesis and relatedendothelial cell functions was examined. α-defensins are found inabundance in human atherosclerotic lesions and are known to modifylipoprotein metabolism and inhibit plasminogen activation. It has beendemonstrated for the first time herein that α-defensins inhibit theadhesion of endothelial cells to extracellular matrix, block endothelialcell proliferation, and abrogate capillary tube formation.

It has been demonstrated that these alterations in endothelial cellfunction caused by α-defensins are mediated through an interaction withECM-associated FN. During angiogenesis, ECM-associated FN isincorporated into an adhesive fibrillar network that regulates diverseendothelial cell functions including growth, differentiation andmigration, by transmitting signals to the cells through specificreceptors, predominantly α5β1-integrin. For this reason, we tested thehypothesis that the interaction between α-defensins and FN mightregulate angiogenesis. In support of this hypothesis, the studiesreported here show that: (i) a5β1-integrin-mediated endothelial celladhesion to and migration towards FN, both under control conditions andunder stimulation by vascular endothelial growth factor (VEGF), wasspecifically inhibited by α-defensins in a dose-dependent manner atconcentrations well below those that are known to be cytotoxic to thiscell type, whereas adhesion and migration to other ECM proteins, such asvitronectin, collagen or fibrinogen/fibrin was unaffected excludingnon-specific cytotoxic effect of defensins. The effect of defensin onthese FM-dependent processes was irreversible over the time coursestudied. (ii) α-defensins completely block VEGF- andsphingosine-1-phosphate-induced proliferation of endothelial cells andinduced apoptosis in a dose-dependent manner. (iii) Capillary-like tubeformation in three-dimensional fibrin-matrices was also inhibited byα-defensins. (iv) Lastly, α-defensins inhibited neovascularization inthe CAM-Assay in vivo.

Although not wishing to be bound by theory, α-defensins may inhibitangiogenesis through several complementary mechanisms in addition todisrupting productive interactions between FN and integrins. Forexample, α-defensins are among the predominant inhibitors of plasminogenactivation at sites of acute inflammation. This may shift theproteolytic balance in and around emerging vascular tissue by decreasingmatrix degradation and remodeling. This would attenuate the capacity ofendothelial cells to migrate and reorganize into capillary tubes as isrequired for effective neovascularization. In addition, it was foundthat α-defensins induce endothelial cell apoptosis and thereby inhibitendothelial cell proliferation. The pro-apoptotic effect of α-defensinsmay be attributed to their anti-adhesive effect or through analternative signal-transducing mechanism.

The physiologic relevance and implications of the observedanti-angiogenic function of α-defensins are several. First, onemechanism by which the innate immune system copes with invasive microbesis to invest them in fibrin, which deprives them of nutrition.α-defensins may contribute to this effect by both inhibiting plasminogenactivation as well as by inhibiting the formation of new vesselsrequired to supply oxygen and nutrients to rapidly dividing organisms.This initial walling off process may be followed by a period of enhancedneovascularization during the healing phase characterized by rubor andedema. The healing phase may also be mediated in part by otheranti-microbial peptides, e.g. cathelcidin peptide LL-37, which isgenerated by monocytes, NK cells, T-cells, B-cells and epithelial cellsand stimulates endothelial cell proliferation and neovascularization viamechanism involving the G-protein coupled formyl peptidereceptor-like 1. Thus, the local balance between pro- andanti-angiogenic peptides may serve a regulatory function thatcontributes to host survival and tissue repair.

Second, the anti-angiogenic properties of α-defensins may extend toseveral pathophysiological processes as well. For example, in additionto impairing the vascular metabolism of Lp(a) and LDL and inhibitingfibrinolysis, α-defensins may also impede the development of afunctional vasa vasorum in atherosclerotic vessels. The hypothesis, thatα-defensins are an endogenous modulator of plaque stability angiogenesisrequires additional study. The presence of α-defensins in human tumorsmay serve a salutary function, helping to control tumor angiogenesis andthereby tumor growth.

Other disorders, e.g. ischemia, secondary to vascular occlusion (clot)or disease (atherosclerosis) may result from unimpeded neutrophilactivation, local release of α-defensin and failure to attain optimalrevascularization. Measures that impede defensin-fibronectininteractions constitute a way to promote natural or therapeutic (e.g.VEGF-) neovascularization.

Taken together, the findings set forth herein suggest that α-defensinsare potent regulators of angiogenesis. α-defensins may thus provide aplatform for developing a novel class of anti-angiogenesis compounds incancer and other conditions, e.g. diverse retinopathies or blindnessassociated with exuberant and pathological vascular growth.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A method of inhibiting adhesion of an endothelial cell tofibronectin, said method comprising contacting fibronectin with anisolated α-defensin in an amount sufficient to inhibit endothelial celladhesion to fibronectin, wherein said α-defensin comprises humanneutrophil peptide 1 (HNP1), HNP2, HNP3, HNP4, human defensin 5 (HD5),HD6, or any combination thereof, thereby inhibiting adhesion of anendothelial cell to fibronectin.
 2. A method of inhibiting migration ofan endothelial cell to fibronectin, said method comprising contactingfibronectin with an isolated α-defensin in an amount sufficient toinhibit migration of an endothelial cell to fibronectin, wherein saidα-defensin comprises HNP1, HNP2, HNP3, HNP4, HD5, HD6, or anycombination thereof thereby inhibiting migration of an endothelial cellto fibronectin.